The present invention is in the area of optical technology in which resolution of an image forming process is improved by reshaping the point spread function of a diffraction-limited spot or line to decrease intensity in its periphery compared to its center, thereby reducing its effective diameter or width. The original examples of this point spread function sculpting technique applied to fluorescence microscopy (Hell, U.S. Pat. No. 5,731,588, Baer, U.S. Pat. No. 5,866,911), now called STED (Stimulated Emission Depletion) microscopy, employed a light pulse wavelength able to induce stimulated emission, and used a doughnut shaped intensity distribution with a central minimum that overlapped with the central maximum of the excitation spot to selectively deexcite those excited fluorescent molecules in the peripheral regions of the scanned spot.
Although this STED technique has been proven in practice and applied to biological studies, where it has demonstrated unprecedented resolution for far-field light microscopy, a major disadvantage is that each fluorescent molecule in the specimen can emit only one photon per reshaping event. To produce the maximum resolution enhancement with the technique, the intensity of each doughnut shaped pulse must be as high as possible, usually many times the energy of a saturating fluorescent excitation pulse. Because with STED, all this energy yields at most one fluorescent emitted photon per molecule, and because only a small minority of emitted photons is detected, to produce acceptable signal levels each pixel must summate the contributions of many excitation/doughnut/measurement events, with a resulting enormous doughnut beam power delivered to the specimen. One consequence is significant photobleaching, which currently is the main limiting factor in the STED technique. The low emission efficiency also results in long required integration times to produce an acceptable signal level, making the technique unsuitable for many biological studies. Furthermore, the STED technique, which in principle can deliver resolution limited only by available doughnut beam power, in practice delivers much more modest resolution gains to avoid specimen and dye damage from the doughnut beam.
One way to increase the fluorescence yield per sculpting pulse is to employ a fluorescent molecule capable of being switched into a non-fluorescent or “off” form by an optical switch over signal, that can be applied specifically to the peripheral parts of the excited spot, and such that following such off switching, the molecule can revert to the “on” state (Hell and Kroug, Appl. Phys. B 60:495(95), Hell, U.S. Pat. No. 7,064,824). If such a switch over signal is applied as a doughnut shaped beam to a spot of molecules in their normal (“on”) fluorescent state, the result will be that a larger fraction of the molecules in the center of the spot will survive the doughnut pulse event in their “on” state than those at the periphery of the spot. Following the doughnut pulse, a surviving fluorescent molecule in the center of the spot will respond to a prolonged fluorescent excitation pulse with repeated excitation/emission cycles, so that it might emit a thousand photons before its fluorescence ends, for example, by photobleaching. Because there is a lower density of “on” fluorescent molecules in the periphery of the spot, the effective diameter of the spot is reduced.
In the original proposal by Hell and Kroug to use this “off-switching” method for microscopy resolution enhancement, the “off” state of the fluorescent molecule was a triplet state, unable to fluoresce. The “on” or normally fluorescing state was switched over to the “off” state by a doughnut shaped pulse of an appropriate wavelength. One problem with the use of the triplet state as the “off” state is that it requires a long dwell time per pixel, prolonging the image collection time. Another problem is that for a given mean lifetime of the triplet state, because of the stochastic nature of spontaneous decay from the triplet state, some of the triplet molecules will decay to the ground state more quickly than the mean, contributing fluorescence in unwanted parts of the spot, and other molecules delay their return, reducing their availability to contribute to subsequent pixels. Another serious problem is that the triplet state can lead to oxygen sensitization, and resulting tissue damage and permanent inactivation of the fluorophore.
The recently reported optically switchable (“photochromic”) fluorescent proteins and dyes offer the possibility of implementation of this off-switching scheme, while avoiding some of the problems of use of the triplet state as the “off” state. Possible dyes include diarylethylene derivatives (Irie, Chem. Rev. 100:1685 (2000)) and (in oxygen-free environments) even the common biological fluorescent dyes Cy5 and Alexa 647 (Heilemann et al J. Am. Chem. Soc., 127:3801 (2005)). However for most biological microscopy applications, the switching proteins related to Green Fluorescent Protein (GFP) currently appear to have the advantage for resolution enhancement. Of these, a protein named Dronpa, derived and mutated from a coral protein (Habuchi et al, Proc. Nat. Acad. Sci 102:9511 (2005)), appears to be the best candidate of all, by far, except for one parameter. Dronpa can be efficiently, repeatedly and completely optically switched back and forth between fluorescent and non-fluorescent states. When in the fluorescent state, it has a very high fluorescence quantum efficiency in physiological environments. As a monomer, it can be incorporated into fusion proteins for labeling of subcellular structures, where it retains its superior optical characteristics, and where its relatively small size and lack of perturbation of structure would make it ideal for superresolution microscopy. It is commercially available, and is the subject of active searching for mutant forms, so it may soon be joined by relatives with different color output, rates of switching and other characteristics. Apart from its use to improve resolution of observation, its switching ability allows it follow cellular protein and organelle trafficking. And by substituting for GFP, it could become a component in the many of the systems developed for sensing local concentrations of ions, second messengers and other cellular parameters (Zhang et al, Nature Reviews 3:906 (2002)), but by allowing resolution enhancement, it could allow such measurements to be carried out with unprecedented spatial resolution.
The one parameter that Dronpa falls short on is light sensitivity to off switching, which is about 1000 times less sensitive than Dronpa's sensitivity to on switching. Dronpa has a quantum efficiency of 37% for on switching (Habuchi et al Proc. Nat. Acad. Sci 102:9511 (2005)), which is similar to the efficiency of a good fluorophore to excitation, and therefore similar to the efficiency of the same fluorophore to stimulated emission deexcitation. Therefore to make up for this reduced sensitivity to off switching, to achieve a comparable resolution enhancement, the doughnut shaped pulse in an off-switching scheme with Dronpa requires about 1000 times the energy of such a doughnut shaped pulse with STED that a good fluorophore requires. Consequently, while with an off-switching scheme, Dronpa can yield about 1000 fluorescent photons per molecule for each doughnut shaped pulse, a thousand fold improvement compared to STED, each doughnut shaped pulse must have 1000 times the energy of a comparable STED doughnut shaped pulse, so the two effects substantially cancel each other out.
The result is substantially no net gain in the number of output photons per doughnut photon compared to STED. Mutants of Dronpa (eg., Dronpa-3) have been developed that are more sensitive to off-switching, but that same increased sensitivity switches the fluorescence off sooner during excitation, decreasing the number of photons per doughnut shaped pulse, at least partly eliminating the benefit of the increased sensitivity. Thus there remains a pressing need for a point spread function sculpting method to allow a switching dye or protein like Dronpa to emit many photons per doughnut pulse, but without requiring substantially more energy per doughnut pulse than STED. Such a method could allow up to a three order of magnitude improvement in the fluorescence out to doughnut power in ratio, and this improvement could translate into an enormous advance in reduction of photobleaching and tissue toxicity, improvement in resolution, and shortening image collection time.